Project PXD001250

PRIDE Assigned Tags:
Biological Dataset

Summary

Title

Cell-type and brain-region resolved mouse brain proteome

Description

To understand the complexity of the brain, connectome and transcriptome maps of high resolution are being generated, but an equivalent catalogue of the brain proteome is lacking. To provide a starting point, we have performed an in-depth proteome analysis of the adult mouse brain, its major regions and cell types, which resulted in the so far largest collection of cell-type resolved protein expression data of the brain. Comparisons of the 12,934 identified proteins in oligodendrocytes, astrocytes, microglia and cortical neurons with deep sequencing data of the transcriptome indicated deep coverage of the proteome. We identified novel protein makers for different cell type and brain regions. These were validated either directly such as in case of cell types or by comparative analysis against Allen mouse brain atlas. The utility and the power of the resource were demonstrated by the identification of Lsamp, an adhesion molecule of the IgLON family, as a negative regulator of myelination in a subpopulation of neurons. Our in-depth proteome analysis of CNS cell types provides a framework towards a system-level understanding of cell type diversity in the CNS and serves as a rich resource to the neuroscience community for the better understanding of brain development and function.

Sample Processing Protocol

Mouse brain/brain regions/acutely isolated or cultured cells were lysed in a buffer consisting of 0.1 M Tris-HCl, pH 8.0, 0.1 M DTT, and 2% SDS at 99°C for 5 min. In case of brain and its regions, the samples were homogenized using bead-beater (MP Biomedicals). The cell lysates were subjected to chloroform methanol precipitation and the proteins were subjected to standard in-solution digestion using LysC and trypsin. We analyzed whole mouse brain and cultured CNS cell types as biological triplicates or quadruplicates after tryptic digestion of lysates and fractionation of resulting peptides using strong anion exchange chromatography. We performed LC-MS/MS analysis with 4hr runs per fraction and higher energy collisional dissociation in a quadrupole Orbitrap mass spectrometer equipped with a high field analyzer. In addition, we analyzed ten brain regions and acutely isolated cell types with our ‘single-shot analysis’ approach. Here, we further optimized this approach which resulted in the detection of more than 11,500 different proteins in only four hours replicate analysis. Such deep proteome coverage in a single run was made possible by matching to the ‘brain proteome library’. Spectra were searched with the Andromeda search engine integrated into MaxQuant.

Data Processing Protocol

Mass spectra were processed using MaxQuant computational platform using versions 1.5.0.8 and 1.3.9.21. The spectra were searched by the Andromeda search engine against the mouse Uniprot sequence database. The search included cysteine carbamidomethylation as a fixed modification and N-acetylation of protein and oxidation of methionine as variable modifications. Peptides with minimum of six amino-acid length were considered and the required FDR was set to 1% at the peptide and protein level. Protein identification required at least one unique or razor peptide per protein group. Quantification in MaxQuant was performed using the label free quantification (LFQ) algorithm. Bioinformatic analysis was performed in the Perseus software environment. All MS data were mapped to gene identifiers obtained from Ensembl for comparison with the RNA-seq data.

Contact

Mario Oroshi, Proteomics
Prof. Dr. Matthias Mann, Dept. Proteomics and Signal Transduction, Max-Planck Institute of Biochemistry, Munich, Germany ( lab head )

Submission Date

21/10/2015

Publication Date

29/10/2015

Publication

    Sharma K, Schmitt S, Bergner CG, Tyanova S, Kannaiyan N, Manrique-Hoyos N, Kongi K, Cantuti L, Hanisch UK, Philips MA, Rossner MJ, Mann M, Simons M. Cell type- and brain region-resolved mouse brain proteome. Nat Neurosci. 2015 Nov 2 PubMed: 26523646